JPH07500911A - Screening methods to identify women at increased risk of premature labor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Background of the invention: Screening method to identify women at increased risk of premature delivery Technical field of invention The present invention relates to a method for detecting impending preterm labor. In detail, the present invention Levels of local inflammation-producing proteins or matrix proteins in cervicovaginal fluid samples Confirming impending preterm delivery by detecting an increase in the Important for increasing survival rate during the neonatal period. In particular, more than half of premature newborns It accounts for as much as 45% of morbidity and mortality in newborns without congenital abnormalities. There is. Premature birth prevention drugs that can delay childbirth were introduced 20 to 30 years ago, but The incidence of term births decreased only slightly. Significantly reduced incidence of premature birth The reason for this failure was a misdiagnosis of early labor and the patient's symptoms indicating premature birth. The disease has progressed too far for preventive drugs to be used and delivery cannot be successfully delayed. It is argued that this is the cause.

Traditional diagnostic methods for preterm labor have high error rates of false negatives and false positives (Friedm an et al., Am, J, Obstet, Gynecol, vol. 104, 544 pages, 1969).

Moreover, traditional methods of confirming preterm labor are limited, especially in patients with clinically intact membranes. Garl et al., 0b stet, Gynecol, vol. 60, p. 297, 1982), or Uterine inversion may occur (Atlay et al., Am, J, 0bstet, Gynecol.

108, p. 933, 1970). Indicates increased risk of premature labor Targeted early biochemical markers were sought.

Recently, Lockwood et al. (New Engl. J. Med., 325 Vol., pp. 669-674, 1991), fetuses in cervical or genu secretions. reported that fibronectin indicates a pregnancy at risk of imminent delivery. The author They found that fetal membranes break down and fetal fibronectin is released into the cervix and vagina. It is claimed that this produces biochemical markers.

Other markers released in the body of women with truly threatened pregnancies should be closely monitored. These women must be screened and their disease stage determined. Can be used to provide additional information about.

Summary of the invention The present invention provides early biochemical indicators that indicate an increased risk of preterm labor. This is what we provide. The method of the present invention involves administering a cervicovaginal sample from a pregnant patient at about 12 weeks of gestation. Obtain a sample of secretions to determine whether local inflammation-producing proteins or matrix proteins are present in the sample. This method consists of measuring the level of protein. The above selected in the sample High levels of protein indicate an increased risk of preterm labor. vinegar. The test method of the present invention is a sensitive and specific screen for at-risk pregnancy. It is possible to detect threatened labor as early as 2 to 3 weeks before delivery.

The test method of the invention is preferably carried out on women who are about 12 weeks pregnant, and at least perinatal period until 37 weeks of gestation, and preferably until delivery if test results are negative. Repeat at each visit (every 2-4 weeks). The test results indicate that the risk of threatened labor is increasing. Testing the patient's fetal fibronectin levels for patients with evidence of It is possible to conduct experiments to confirm the increased risk and estimate how early labor may be. can. Moreover, these patients should be treated with the same care as any other patient at risk. Can be carefully monitored.

Detailed description of the invention The present invention provides a screen that provides early biochemical indicators of an increased risk of threatened labor. This is a testing method. The method of the present invention allows for the prevention of preterm labor as early as 2 to 3 weeks before delivery. can provide indicators. The method of the present invention allows for early intervention in the process of preterm birth. can show that these pregnancies are at greatest risk. Additional factors that can be used can be provided.

The method of the present invention can be used from about 12 weeks after pregnancy to less than about 36 or 37 weeks of pregnancy. Obtain a sample of cervicovaginal secretions from the vaginal cavity or external cervix of a pregnant patient, and to measure the levels of local inflammatory proteins or matrix proteins. This method consists of The protein is present at high levels in the sample. indicates patients at risk of premature delivery.

By the method of the present invention, from early pregnancy until delivery at 36 or 37 weeks, Delivery can be confirmed. Delivery before 20 weeks of gestation is natural rather than premature. It is called birth. The method of the invention covers spontaneous abortion (12-20 weeks gestation) and It can be used to detect premature birth (20-37 weeks gestation).

Patient to be tested The method of the present invention extends from approximately 12 weeks of gestation to the expected gestational age (36 or 37 weeks). Can be used by any pregnant woman. screening pregnant women screening to determine if labor is imminent. Patients should include those with clinically intact membranes who are at high risk for preterm delivery. and preferably those whose pregnancy is not sufficiently advanced to ensure delivery of a healthy fetus. All women. 90% of fetal morbidity and 100% of fetal deaths associated with preterm birth are caused by pregnancy. A fetus delivered at less than 20 weeks of gestation. Therefore, a 32-34 week pregnancy is a fetus. This is an important health deadline for women whose gestational age is at least about 12 weeks and less than 34 weeks. Women must be tested.

Additionally, there are a number of known factors associated with the risk of preterm birth. As these factors multiple fetuses; incomplete cervix; uterine abnormalities; polyhydramnios; nulliparity; Stage rupture or early labor pre-dementia; vaginal bleeding during the first 3 months; antenatal nursing care Little or no pain; as well as abdominal pain, low back pain, and cervical mucosa. excretion and contraction. Gestational age >12 weeks with clinically intact membranes pregnant women with one or more risk factors for preterm delivery during the risk period, i.e., about 3 Must be tested from 4 to 37 weeks.

sample Samples are obtained near the radial fornix, at the genu of the uterus, or at the external cervical opening. The sample is one Generally composed of fluid and particulate solids, containing the mucous membranes of the vagina or cervix; the remainder is the secretion of the cervix. The sample is a swab with a fibrous tip such as Dacron. It is preferable to take it out.

Alternatively, the sample can be obtained with a suction or washing device. The collected sample is The calculations to be made for further dilutions using the assay procedure are described in the section describing the assay procedure. You can do it in the same way as you do.

After collection, the sample is transferred to a suitable container for storage and transported to the testing laboratory.

It is important that the sample is dispersed in a liquid that preserves the protein analyte. . The storage and transportation media should minimize the loss of analyte levels during storage and transportation. and preferably prevent it. Suitable solutions for storage and transportation are: 0.05M Tris-HCf, 1) H7,4; 0.15M NaCl! ; 0. 02% NaN, 1% BSA, 500 recrein units/ml aprotinin ;1mM phenylmethylsulfonyl fluoride (PMSF) and 5mM EDT U.S. Patent No. 4.919.88, issued April 24, 1990, consisting of A. It is stated in No. 9. This solution is also suitable as a sample dilution solution.

Alternatively, home and clinic equipment may be used to process samples immediately. . If used, the sample should be placed directly into the device described above and within minutes of collecting the sample. Conduct the test. In such cases, the need to stabilize the analyte is minimal and the assay Any solution that is easy to carry out and is harmless to the stability of the analyte can be used.

Test object local inflammatory proteins Local inflammation-produced proteins are synthesized and secreted during acute inflammation, and are secreted by cells. It has a protective effect that helps maintain biological homeostasis in the extravesicular space. this station Inflammatory proteins are known and are involved in complement-activated cellular and humoral immune responses. There are proteins produced by

There are five local inflammatory proteins that are released as part of the local inflammatory response. There are major types of proteins. Cytokines are the primary source of local inflammatory proteins. This is the first category. As cytokines, interleukins (especially IL-1 , IL-6 and IL-8), transforming proliferators (particularly TGFβ) and and tumor necrosis factors (TNFα and TNFβ). Prostanoids are local inflammation This is the second category of disease-producing proteins. As a prostanoid, prostagra There are leukotrienes and leukotrienes. However, prostaglandins are present at 20 weeks of pregnancy. It is preferable to test later. The third category of local inflammation-producing proteins involves an acute conflict. Contains responsive proteins. Fibronectin is an acute phase reactive protein. celluloblasmin, C-reactive protein, C1-anti-trypsin, C1- Anti-chymotrypsin, α, -macroglobulin, interferons, fibril serum amyloid A protein, complement (particularly C3 and C4) and C1-acid sugars. It has protein. The fourth component is locally responsive immunoglobulin 11? A and 1gM is included. The fifth category includes soluble factors derived from macrophages and monocytes, Contains toferin and lysozyme.

These local inflammatory productions are known to be produced in reproductive organs and connective tissues. Protein is important.

extracellular matrix proteins A significant portion of tissue volume is extracellular space; It is filled with a complex network of macromolecules consisting of an outer matrix. this cell The external matrix consists of locally secreted proteins that determine the physical properties of the tissue. Composed of polysaccharides.

As used in this application, the term "extracellular matrix protein" refers to the selected Refers to the adult isoform of extracellular matrix protein and fetal fibronectin. It does not include fetal isoforms of proteins present during pregnancy, such as preferred extracellular Matrix proteins are present in the amniotic epithelium, serosa, uterine decidua epithelium, and basal lamina. This is the protein. Further, the protein is preferably inflammatory or proteinaceous. the maternal/uterine decidua interface and/or cervix as a result of the process of protein decomposition. proteins released from the maternal/uterine decidual interface and/or is excessive production in the cervix stimulated by abnormal autocrine events or present as a result of secretion. In addition to detecting extracellular matrix proteins , produced by proteolytic cleavage, including chemical and enzymatic cleavage of proteins. They also plan to detect the resulting protein fragments. these proteins The fragment is a degradation product of the protein, and the fragment is a degradation product of the protein. Contains the portion of carbohydrates that are released when

There are two functional types of extracellular matrix proteins: There are proteins and adhesive proteins. In addition, extracellular 71 helix protein contains secreted proteins that are secreted in large quantities by cells near the site. cell Structural proteins of the outer matrix include collagen and elastin. cell Adhesive proteins in the outer matrix include fibronectin, tenascin, and bicarbonate. There are tronectin and laminin. The secreted protein includes insulin. growth factor binding protein (IGPBP) (especially IGFBP-1), Elas Chin receptor protein, troboelastin, trobocollagen, decorin (del matan sulfate), mucin and CA125. Degradation production of these proteins Examples include desmosine (a breakdown product of elastin), sialic acid (many of these carbohydrate moieties released by protein breakdown) and peptide flags There is a ment.

Certification procedure Selected local inflammation-producing proteins or extracellular matrix proteins are assayed using a procedure that confirms the presence of a selected protein in a threshold amount in the sample. be done. Immunoassays are preferred. Proteins (such as The affinity of the antibody required to detect the analyte of the assay is different from that of other polypeptides. The affinity of the antibody required to detect the analyte is no different. even more , selected local inflammatory proteins or extracellular matrix proteins This assay is not possible if the protein undergoes proteolytic cleavage. That is, these tags Many epitopes of proteins, especially large extracellular matrix proteins, are This is because it remains intact even after protein degradation. In such cases, antibodies against the epitope does not distinguish between intact forms of proteins and protein fragments. But A proteolytic reaction removes the epitope recognized by the selected antibody In this case, antibodies that bind to epitopes that remain intact can be selected. .

In some cases, proteolytic reactions may cause , may create an epi-I-b that is present only in the generated fragment. mosquito in such cases, to detect the generated fragments, The use of specific antibodies is also contemplated by the present invention.

Additionally, confirmation of the presence of extracellular matrix protein fragments Certification will also be conducted. Proteolytic reactions remove enzymes that are not present in the intact protein. If a pitope is generated, the presence of a threshold amount of fragments indicates that the pittope is generated. It can be detected using an antibody against the pitope. Additionally, one or more epi If the tope is intact, measure the size of the protein bound by the selected antibody. Detect the presence of fragments of the protein using a Western blot assay It can be tested.

Anti-analyte antibodies can be made in many ways. Polyclonal antibodies are selected administering to a host animal an immunogenic composition containing a local inflammatory protein produced by It can be induced by

Does the method of manufacturing immunogenic compositions of proteins vary depending on the host animal and protein? One is publicly known. For example, the test protein or its antigenic portion may be KLH or BSA. can be conjugated to immunogenic substances such as or in adjuvants etc. can be provided to The elicited antibodies are tested and the composition is tested against the protein being tested. You can check whether it corresponds to the quality. Polyclonal antibody composition desired If the antibody does not provide specificity, it can be purified and characterized by a variety of conventional methods. It can enhance the opposite sex. For example, the composition may include an analyte immobilized on a solid support. It can be purified by contact with proteins to reduce its binding with other substances. can. Those antibodies that bind to the carrier are retained. Sephadex, Cephalo of various solids such as affinity chromatography materials, including Purification methods using antigens immobilized on carriers are known.

Monoclonal analyte-specific antibodies can also be produced by conventional methods. Subject Inject an immunogenic composition containing the protein into a mouse and then obtain pancreatic cells. . These pancreatic cells can be fused with a fusion partner to create hybridomas. can. The antibodies secreted by this hybridoma are screened and reacts with the analyte and substantially indicates a reaction with other proteins present in the sample. It is possible to select hybridomas that do not. Produce antibodies of desired specificity Hybridomas are cultured using standard methods. Hybridoma production and culture methods are publicly available. methods known in the art and do not form any part of the invention.

A typical method for producing polyclonal anti-fibronectin antibodies is described in the Examples. Ru. Antibody production and purification methods are described in numerous publications and use a variety of protocols and labels. A number of different immunoassays are known. The assay conditions and reagents are based on conventional technology. It can be of any type found. The test method is heterogeneous test or The homogeneous test method may be used, but the simplest method is the sandwich test method.

The assay typically utilizes an anti-analyte antibody immobilized on a solid phase.

The antibody may be polyclonal or monoclonal. fixed in its solid phase The antibody is then bound to the sample. This antibody-sample binding can be confirmed in a number of ways. I can do it. Complex generation involves the use of soluble antibodies specific for the analyte protein. This can be confirmed by These antibodies can be directly labeled or can be detected using a labeled second antibody specific for the soluble antibody species. .

Various labels include radionuclides, enzymes, fluorophores, and colloidal metals. flight A useful assay is quantitative enzyme-linked immunosorbent assay (ELISA). In this method, antibodies specific to the test protein are immobilized on a solid phase and fermented. It is used as a naturally labeled soluble antibody. Alternatively, this assay can be used for competitive inhibition. This can be done based on. In that case, the analyte in the sample is a predetermined amount of anti-analyte. The antibody is competed with a known amount of analyte or analyte analog. For example fib When assaying for ronectin, all fibronectin present in the sample is A known amount of labeled fibronectin or fibrin is added to the binding site of Can compete with Ronectin analogs.

and of labeled fibronectin immobilized on the solid phase or remaining in solution. Amounts can be measured.

In another preferred embodiment, the assay is a homogeneous immunoassay. In this method, antibodies specific to the analyte are immobilized on a solid phase and colloidal metal used as a soluble antibody labeled with By diluting the conjugate appropriately Therefore, the analyte at the selected threshold level can be detected as a positive sample.

threshold The amount of local inflammation-producing proteins or matrix proteins in a sample is Significantly increased above the level of pregnant women who are in the gestation stage and have a normal pregnancy. are doing. The selected threshold value is preferably a value of normal value + 2 x standard deviation.

Quantifying proteins in vaginal swab samples is difficult for two reasons. first The reason for this is that the amount of body fluid collected with the swab varies. And the second reason is a variation in the total volume of secretions present in the cervicopupil region. Therefore, especially All measurements of protein concentration in a given sample are based on protein concentration within the cervicovaginal region. It is only a semi-quantitative indicator of the total amount. Therefore, patients whose samples show values close to the threshold It is advisable to retest at the follow-up visit.

Local inflammation-producing or matrix proteins selected for analysis The threshold (threshold associated with the risk of preterm delivery) varies. Moreover, this threshold is May vary depending on patient's stage of pregnancy.

Determine thresholds for specific local inflammatory proteins or matrix proteins. A prospective-retrospective study was conducted to determine specific 5study) to detect one or more selected local inflammatory productions. Determine the threshold for raw protein and/or matrix protein.

These tests are known and can be performed in a number of ways. representative preference The new method is described below.

In a preferred study, at least 10 samples from each of approximately 2,000 pregnant women will be used. get the fee. Sample collection begins at approximately 8 weeks of gestation and is performed at equal time intervals as desired. cormorant. Samples are collected at least every 3-4 weeks. Samples should be collected every two weeks is preferred.

Approximately 5-10% of pregnant women go into preterm labor and separate early without premature rupture of membranes. Give birth. Therefore, of the 2,000 women in this study who delivered early Approximately 50 women who do will go into preterm labor without premature rupture of the membranes.

When identifying these women, select matched controls. These controls were age , harmonized on known risk factors such as menstrual bleeding, parity history and socio-economic factors. let Samples from controls are then matched for sample time and stage of pregnancy. Next Measure the levels of local inflammation-producing proteins and matrix proteins on the sample. Set.

Next, the receiver-operator statistical method (receiver-operator statistical method) is used to measure sensitivity and specificity. 5 statistics). This statistical method is at risk. Only a small number of women tested are false positives, which is a satisfactory level of false positives. It can also be used selectively for screening to obtain the same results. The threshold is The system can be adjusted to detect virtually all women at risk. preferable. This is because other criteria should be considered to further reduce the risk before starting treatment. This is because it can be evaluated.

600-750 ng/ml range for samples from women from approximately 20 weeks of pregnancy until delivery Tests showing that a threshold value of total fibronectin is indicative of danger are It is described in detail in the Examples.

Explanation of test results High levels of local inflammatory proteins or matrix proteins are associated with early labor. Indicates increased risk. As detailed in the examples, the total fi The assay for bronectin is sensitive and specific. Moreover, this test method High negative predictive value. It is believed that a high percentage of patients who delivered early have fibrone This means that the cutin level was high. The test method of the present invention is suitable for patients who give birth early. Since a large percentage of patients are successfully detected, other indicators of risk may be completely absent. It is an effective screening method for women at risk of premature labor.

The test method of the present invention extends from about 12 weeks of gestation until delivery, or at least early delivery. It is recommended for all pregnant women until the risk of delivery is no longer present (i.e. until approximately 8 weeks of pregnancy). can be administered. The test method of the present invention applies to known risks from the 12th week of pregnancy until delivery. It is recommended that all women with the factor be given this treatment.

Positive test results for local inflammatory proteins or matrix proteins (if above the threshold), the patient has fetal fibronectin in their cervicovaginal secretions. It is preferred to test for the presence of chin. Fetal fibronectin is secreted above If present in the body, the patient is likely to go into labor after 2-3 days.

Measures to confirm or increase fetal lung maturity may also be used. fetal fibronec If the Chin assay result is negative, the patient should be closely monitored and the patient's fetal fibrosis Assessment of nectin levels should be repeated at subsequent visits. one In general, testing of patients at risk of preterm labor is limited to testing patients who are in the low-risk category. It is performed every 2 weeks between about 22 and 36 weeks of pregnancy, unlike every 4 weeks in the case of Ru.

Negative test results for local inflammatory proteins or matrix proteins , the test will be performed at each subsequent antenatal visit if the test result is positive or the patient It is preferable to repeat this until the expected delivery date is reached.

The invention will be further illustrated by, but not limited to, the following specific examples. It's not something you can do. Unless otherwise noted, temperatures are in degrees Celsius and concentrations are in weight percent. show. Procedures presumed to be performed should be written in the present tense and should be performed in the laboratory. The steps are written in the past tense.

Example 1 Quantification of total fibronectin in vaginal swab samples.

The following reagents and procedures were used for an immunoassay to measure total fibronectin in vaginal samples. used.

Production of polyclonal anti-human fibronectin antibodies Human plasma fibronectin , Engvall and RuoslaMi, Int, J.

Cancer, Vol. 20, pp. 1-5, 1977. It was purified from human plasma. For example, in the literature, 5tollar, Meth, Enz)a +, vol. 70, p. 7n, 1980, the immunization method and its plan. The anti-human plasma fibronectin antigen was used to immunize goats with the human plasma fibronectin antigen described above. Fibronectin antibodies were induced in goats. Antiserum is a place for monoclonal antibodies. Solid phase assays similar to those used in the present invention, such as Lange et al., Cl1n.

T! xp, Immunol, vol. 25, p. 191, 1976 and Pis etsky et al., J. Immun.

Methods such as those described in Meth, Volume 41, Page 187, 1981. Screened with.

The IgG fraction of the above antiserum was further subjected to affinity chromatography as follows. It was purified with In other words, the method recommended by the manufacturer (AFFINITY CHROM ATOGRAPHY, Pharmacia Fine Chemicals C human plasma fibrone according to Atalogue 1990, pp. 15-18). CNBr-SephaC7-su4B coupled with cutin (Pharma Affinity chromatography using CIA Fine Chemicals It is a fee.

Fill the above column with 2-3 volumes of buffer (0.01M PBSSpl(7,2),! : Equilibrate briefly, then apply solution containing anti-human fibronectin antibody to the column. added to. absorbance of the eluate until no more protein flows out of the column. was monitored at 280 nm. Next, the baseline absorbance at 280 nm was obtained. The column was washed with equilibration buffer until the column was washed with equilibration buffer.

Immune affinity captured anti-human plasma fibronectin antibody was 0.1 M It was eluted with glycine buffer pH 2.5. Collect and pool peak protein fractions. 0. For OIM PBS pH 7.2, change the buffer many times and bring it to 4℃. and dialyzed for 24 to 36 hours.

Rabbits were immunized with human plasma fibronectin by repeating the above method, and the obtained A polyclonal anti-human fibronectin antibody was purified.

Preparation of microtiter plates coated with anti-fibronectin antibodies as described above. The produced goat anti-human plasma fibronectin was added to 0.05M carbonate buffer pH 9.6. It was diluted to 10μg/ml. 100 μf of this diluted solution was obtained from Co5tar Co., Ltd. Polystyrene traces such as those supplied by Nunc or Dynatech Distributed into each well of the titration plate. Cover the plate and let it sit at room temperature for 2-4 minutes. Incubate for 1 hour or overnight at 4°C. Wash that plate Buff er (0,02M) Squirrel HC4,0,015M NaC1,0,05% Tweet Wash 3 to 4 times with 1-20 g of water, fill the wells before each use, and then empty the wells completely. Make it. Next, add blocking/stabilizing solution (4% sucrose) to each well of the plate. S, 1% mannitol, O, OIM PBS, 1% BSA, 0.02% NaN5 5 pH 7,4) Block by dispersing 200 μl each, and store at room temperature. Incubate for 30 minutes to 2 hours. The wells are then vacuumed dry and the plate , packaged in an airtight container containing a desiccant pouch and stored at 4°C until needed. Ta. These wells are prepared as strips of 8 wells as described above. The anti-human plasma fibronectin antibody was prepared by Avrameas, [mmunoch em, vol. 6, p. 43, 1969 one-step glutaraldehyde method. Then, it was conjugated with alkaline phosphatase.

Assay reagents The assay was performed using the following additional reagents: The raw antibody conjugate was diluted with conjugate diluent (0, 05M) Lys buffer, pH 7, 2, 2% D-sorbitol, 2% BSA, 0.1 % sodium azide, 0.01% Tween-20, 1mM magnesium chloride and (and 0.1% zinc chloride) and add 10 ml to a polyethylene dropper. I put it in.

The substrate for the enzyme (10 mJ' in a polyethylene dropper container) was 0,4M aminomethyl protein containing magnesium and 0.2% sodium azide Phenolphthalein monophosphate dissolved in lopanediol buffer pH 10 It was (1■/-).

Fibronectin calibration solution is sample dilution solution (0.05M) Liss buffer pH 7 , 4.1% Bovine Serum Albumin (BSA), 0.15M Sodium Chloride, 0.0 2% sodium azide, 5mM ethylenediaminetetraacetic acid (EDTA), 1 　mM phenylmethylsulfonyl fluoride (PMSF) and 500 force recrein unit/-aborotinin], 0.0, 0.01, 0.05 and 0.25 μg Plasma fibronectin (Ind., Indiana, USA) diluted to a concentration of Humans purchased from Boehringer Mannheim, Dianapolis Serum-derived fibronectin; catalog number 1050407). this The sample dilution solution is described in the U.S. patent of Jones et al., issued April 24, 1990. No. 4.919.889. This patent is incorporated in its entirety into this application. It is something that Negative controls were used without fibronectin as positive controls. It was a sample dilution solution.

The rinse buffer (10 mJ in a polyethylene dropper bottle) was 1.0 M Tris buffer p H7,4,4,0M sodium chloride, 2.5% Tween-2O and 1% sodium azide It was a 50-fold concentrate containing lium. This rinse buffer is used for the assay. 0.02M) Squirrel, 0.08M Sodium Chloride, 0.05% Tween- Diluted with water to a final concentration of 20 and 0.02% sodium azide.

In addition, a polyethylene sample filter with a pore size of 5 μm (Louisiana, USA) was used. (Porex Technologies, Inc., Fairburn, NA). The samples were filtered before assaying. All drop bottles used to perform this test are in a polyethylene bottle designed to add approximately 50 μl droplets of reagent. Ru. All assay steps following sample collection use the reagents and materials listed above. did.

Certification procedure The test was conducted as follows. All samples were taken from the posterior vaginal circle using a Dacron swab. Collected near the operculum or cervical opening. Collect the swab sample in a vial Sample diluent in 1. (Immersed in W.

The swab was removed from the solution, leaving as much liquid as possible in the collection tube. child These samples were incubated with controls for 15 minutes before running the assay, before or after filtration. Incubated at 37°C. Place the sample filter in the correct position on each sample tube. It has a snap closure. Place the 8-well strip in the appropriate strip holder. It snapped into place. This holder holds 12 standard microtitration plates. It has an alphanumeric display with columns and eight rows. Two 100μl samples and positive The sex and negative controls were placed in separate wells of the microtiter strip and incubated for 1 hour at room temperature. Incubated.

Following incubation, samples and controls were aspirated from the wells.

Wells were washed three times with diluted wash buffer (lx). After washing, enzyme-antibody Zygote 100μ! was added to each well and incubated for 30 minutes at room temperature. So The wells were aspirated and washed as described above. Washing followed by enzyme substrate 10 0 μβ was added to each well and incubated for 30 minutes at room temperature.

After incubation, gently shake the plate by hand or on an orbital shaker. The contents of the wells were mixed by shaking. ELISA plate the frame of the strip. into the card reader. The absorbance of each well at a wavelength of 550 nm was measured. each The average absorbance of duplicate sample and control wells was calculated. Total fibrone of the sample To determine the concentration of Chin, prepare a standard curve and estimate that the sample has been diluted to approximately 1/lo of the original concentration. (Collect approximately 0.1-yd sample and mix with 1.0 yd diluent).

Example 2 Detection of threshold amounts of total fibronectin in vaginal swab samples Another preferred embodiment The assay kit for detecting the threshold amount of fibronectin contains the following elements: Ru. This kit is suitable for rapid assays or for assays at your doctor's office. It is designed for use in

1. Has a plastic housing and (a) anti-fibronectin antibody captures Porous nylon membrane: (b) a flow rate control membrane system; and (c) absorbent layer; Verification device containing 2. Goat anti-fibronec labeled with colloidal gold in protein matrix Chin antibody conjugate, 3. Buffer for reconstituting the conjugate; 4. Washing solution; 5. Sterile Dacron sample collection swab.

The membrane device is manufactured using the following steps. Prepared similarly as described in Example 1. Approximately 2 μl of polyclonal anti-fibronectin antibody was added to pH 6,0,01MIJ :/Acid Buffered Saline (PBS), 0.1M containing 0.5trg/ml BSA The surface of the membrane (1,2μ nylon, Biodyne-A, Apply to Pal l). Semen as described in Example 1 in the same buffer. A procedural control consisting of purified human plasma fibronectin was also applied to another area of the membrane. Ru. After air-drying the membrane, a buffer consisting of 0.5% nonfat dry milk buffered with PBS was added. Add locking reagent to membrane. Remove excess blocking reagent for at least approximately 20 minutes. Remove later.

To control the flow rate of sample solution from the assay membrane to the absorbent layer, a membrane support device (Targ et Device, 'V-Tech, Bomona, California, USA) , a second porous layer (0.45μ low protein Assembled with quality bonded nylon, LoPro-dyne, Pa1l). Next 2 The porous membrane has a volume of more than 1.5 mj'. Place the layer (Chromex Inc., Plutzklin, New York, USA) above. into the device. The device is placed inside a sealed plastic bag containing desiccant. individually packaged.

Colloidal gold is produced using a method that produces particles of approximately 30 nm. Prepared by reducing the acid with 0.16% sodium citrate. The above membrane Separately heat the solution briefly at 90°C. Then, while stirring vigorously, the above reduction is carried out. a solution of the membrane is added to the membrane solution. Boil the mixed solution for at least 10 minutes (100″C).

Affinity-purified goat anti-fibronectin antibody (as described in Example 1) (prepared in this way) was captured on colloidal gold by adsorption. The above manufactured co Lloyd's gold was easily conjugated with antibodies (5-10 μg/yd) in water. Following joining The conjugate solution was supplemented with 5% BSA and 5% polyvinylpyrrolidone (final concentration). It is stabilized by adding

The raw conjugate can be obtained by ultrafiltration using a hollow fiber filter. It was concentrated 12 times. The resulting enriched conjugate was dissolved in 15mM) Lis, 2% BSA, 0.1 %Tween 20.0.2% polyethylene glycol, 8% polyvinylpyrrolidone and diluted to appropriate levels with 0.04% thimerosal. The appropriate concentration is below Use the range of dilutions for the sample assay described above and check the dilution that gives the best results. Determine by This titration method has a detection threshold for total fibronectin. Use with the bell set to 750ng/+ni.

The selected conjugate dilutions are placed in polyethylene sample collection tubes and lyophilized. child During freeze-drying, the collection tube was fitted with a polyethylene sample filter with a pore size of 2μ. - (Porex Technology, Fairburn, Georgia, USA) Company S) is installed. Freeze-dried zygotes are individually wrapped in foil with desiccant Packaged in a pouch.

The buffer used for conjugate reconstitution is 100mM sodium acetate.

This buffer is packaged as single doses in 1 ml disposable test tubes. cleaning solution The liquid is water and is packaged as single doses in disposable test tubes. The kit of the present invention Also includes a separately wrapped Dacron sterile swab and a card summarizing the procedure. ing.

The test was conducted as follows.

1. Before collecting the sample, wrap the plastic test tube containing the whole zygote in foil. Remove from pouch, remove dropper tip, and add buffer to reconstitute the conjugate. Add all contents in the test.

2. Collect the sample with the swab provided. After the vagina during inspection with sterile specula Insert the swab into the sunshade and rotate for approximately 10 seconds to absorb body fluids. Try it now Start the experiment. Samples should not be stored and tested after a period of time has elapsed. that swab Place in total conjugate solution and mix rapidly by up and down motion for 10-15 minutes.

3. From the swab by rotating the swab tip over the inside of the test tube Only remove large amounts of liquid. Acceptable methods when handling potentially infectious materials Dispose of the swab.

4. Return the dropper tip to the plastic test tube and immediately remove the diluted and filtered sample. Dispense the entire volume of the sample onto the surface of the membrane device.

5. After absorbing the sample solution onto the membrane surface, add a few drops of cleaning solution and observe the result.

6. Negative test results are indicated in red in the control area for this method of membrane only. positive The test result is a pink or red spot on the test area of the membrane as well as the control area. It is indicated by.

Example 3 Total fibronectin levels in patients with early labor.

Total fibronectin expressed in the cervix as a screen for early delivery In an effort to evaluate, 73 asymptomatic women at high risk of premature delivery were identified and followed up. I had a inspection. Factors that defined these women as being at high risk included twin pregnancy; If you have a history of pregnancy, uterine abnormalities, preterm labor, or early delivery. Are these women? Vaginal specimens were obtained at 1-2 week intervals between 24 and 34 weeks of gestation. external cervical dilatation Remove vaginal secretions from the oral cervicovaginal area and the posterior fornix of the vagina with a separate Dacron swab. collected using.

Total fibronectin concentrations in vaginal secretion samples for this study were as described in Example 1. Measurements were made in the same manner. The average concentration of total fibronectin in uterine vaginal secretions is 4,49 ± 0.50 μg/d (average) for uncomplicated pregnant women less than 22 weeks pregnant. Mean value±SEM). The total fibronectin concentration was 64.8 in these women. % (250/386) exceeded 750 ng/rd. Total after 22 weeks of pregnancy The average concentration of fibronectin was 0.99±0.50. and 22.2% (4/18) was larger than 7501 g/-.

From these values, the sample has a total fibronetatin concentration of more than 750 ng/d. The test result was determined to be positive. If a single sample is positive, the patient is positive It was the minimum necessary condition to define. Accumulate sample results for all women at delivery compared to gestational age. Delivery before 37 weeks is defined as early delivery, and delivery after 37 weeks is defined as early delivery. A full-month pregnancy was defined as a full-month pregnancy.

Of the 73 patients enrolled in this evaluation, 24 delivered premature babies and 49 delivered full-term babies. I gave birth. Table 1 shows the relationship between the measurement results of total fibronectin and clinical results.

Sensitivity in these analyzes refers to the number of true positive test results The number of true positive test results divided by the total number of women in the false negative test results divided by the total number of negative and false negative test results. Singular means true The number of negative test results divided by the total number of women without the above symptoms, i.e. the true The number of negative test results divided by the total number of true negative test results and false positive test results means value. Positive predictive value (PV+) is the number of true positive test results that indicate a positive means the value divided by the total number of samples. Negative predictive value (PV-) is the true negative means the number of sex test results divided by the total number of samples showing negative results. sensitivity, Specificity, positive predictive value, and negative predictive value are It is based on the detection of preterm labor (PTD) rather than the detection of preterm labor (PTD).

Mantel-Haenszel L=5.98, p<0.014 Parturition at premature birth At least one total fibronectin sample from 19 of 24 patients with 7 s on/- (specificity 82.8%). Among the 49 patients who gave birth during the full moon, At least one sample of total fibronectin from 24 people was 750 ng/lnI! (specificity = 51.0%). Alternatively, the predictive value of a positive test is 44.2%. The predictive value of the negative test was 83.3%. The relative risk associated with a positive test (negative ) was 2.65, which has a p-value of less than 0.007. is significantly different from 1.00 (zero hypothesis (Ho): relative risk -1,00). Ru. Furthermore, analysis of positive test results was performed using the Mantel-Haenszel L test standard. It is associated with early delivery as shown by the total value of 5.98 (p < 0.014). It was.

The test results show that the above assay is effective for most women who give birth early. The screening assay was sensitive and specific in that it detected It shows.

Example 4 Total fibronectin levels at 12-22 weeks of pregnancy.

The cases described in Example 1 were obtained from women with no apparent complications between the 12th week of pregnancy and the due date of delivery. Measure the concentration of total fibronectin in a sample of cervicovaginal secretion obtained as described above. Established. The mean concentration of total fibronectin was determined at each gestational age.

Total fibronectin concentration is highest at 12 weeks of pregnancy and gradually decreases until 22 weeks of pregnancy. Ta. From 22 weeks of gestation until the day of delivery, total fibronectin concentrations rarely drop below 5. It did not exceed 00 ng/ml. When gestational age exceeds 34 weeks, total fibronectic The concentration gradually increased and predicted parturition.

The gestational age dependence of total fibronectin concentration was determined by the estimated gestational age at the time of sampling and total fibronectin concentration. Calculated using fibronectin concentration as independent and dependent variable, respectively. It was accurately predicted by a series of second-order polynomial regression equations.

The predicted concentration of total fibronectin (μg/d) at each gestational week is shown in the table below. .

Gestation age Predicted concentration [total FN) Gestation age Predicted concentration [total FN) 12 6.30 2 5 0.10 13 6, 20 26 0.06 14 5, 94 27 0.03 15 5, 51 28 0.04 16 4.92’ 29 0.07 17 4.16 30 0.13 18 3.24 31 0.22 19 2.16 32 0.33 20 0, 91 33 0.47 21 0, 57 34 0.64 22 0, 42 35 0.83 23 0.29 36 1.06 24 0.18 37 1.31 38 1, 58 Submission of translation of written amendment (Article 184-8 of the Patent Act) , month λ day in 1994

Claims (21)

[Claims] 1. (a) From a pregnant patient after 12 weeks of pregnancy, from the vaginal cavity or cervix. Obtain a secretion sample; then (b) Cytokine, leukotriene, collagenase, plasminogen activity factor, acute phase reactive protein, IgA, 1gM, macrophages and monocytes Soluble factors, lactoferrin, lysozyme and extracellular matrix proteins from For samples of proteins selected from the group consisting of proteins or their degradation products. measuring the level of The presence of high levels of said protein in the sample indicates an increased risk of calving. Check for early indicators that indicate an increased risk of preterm labor. How to do it. 2. The proteins include interleukins, transforming growth factors and The method according to claim 1, wherein the cytokine is selected from the group consisting of tumor necrosis factor. . 3. The protein may include fibronectin, ceruloplasmin, α1-anti-avian psin, α1-anti-chymotrypsin, α2-macroglobulin interferons , fibrin, serum amyloid A protein, complement and α1-acid glycoprotein The method according to claim 1, wherein the acute phase reactive protein is selected from the group consisting of . 4. the protein is an extracellular matrix protein; 2. The method according to claim 1, wherein the adhesive protein is an adhesive protein. 5. The adhesive proteins include fibronectin, tenascin, vitronectin, and 5. The method according to claim 4, wherein the method is selected from the group consisting of: and laminin. 6. the protein is an extracellular matrix protein; 2. The method of claim 1, wherein the protein is a structural protein. 7. the structural protein is selected from the group consisting of collagen and elastin; 7. The method according to claim 6. 8. the protein is an extracellular matrix protein; 2. The method of claim 1, wherein the protein is a secreted protein. 9. The secreted protein is insulin-like growth factor binding protein, elastin receptor protein, tropoelastin, tropocollagen, decorin, mucin and CA125. 10. Claim that the protein is a degradation product of an extracellular matrix protein. The method described in Section 1. 11. wherein said decomposition product is selected from the group consisting of desmosine and sialic acid; The method according to claim 10. 12. 11. The method of claim 10, wherein the degradation products are peptide fragments. 13. 2. The method of claim 1, wherein the sample is removed from the retrovaginal fornix. 14. 2. The method of claim 1, wherein the sample is obtained from the cervical opening. 15. Because the sample obtained from the patient does not contain high levels of said protein. After at least two weeks, another sample from the patient is assayed for the protein. 2. The method according to claim 1. 16. Obtain a sample from the patient and use the sample until the patient delivers or if a positive sample is found. 2. The protein is assayed for the presence of said protein at intervals of about 4 weeks until obtained. 5. The method described in 5. 17. Since the sample contains high levels of the protein, 2. The method of claim 1, wherein the sample is assayed for the presence of fetal fibronectin. 18. At least 2 weeks since the sample does not contain fetal fibronectin. 2. After that time, another sample from the patient is assayed for fetal fibronectin. 7. The method described in 7. 19. Obtain a sample from the patient and use the sample to determine whether the patient delivers or has a positive fetal Presence of fetal fibronectin at 2 week intervals until fibronectin samples are obtained 19. The method according to claim 18, wherein the method is assayed for. 20. (a) A sample is subjected to antigen-antibody binding with an antibody specific to the protein. (b) measuring the amount of binding; The method described in Section 1. 21. (a) Secretion sample from the vaginal cavity or cervix from a pregnant patient after 20 weeks of pregnancy obtain; then (b) After 20 weeks of pregnancy, measure the level of prostaglandin in the sample; Ranari: The presence of high levels of these prostaglandins in the sample increases the risk of delivery. early indicators of increased risk of preterm labor How to check.